One obstacle to the robust production and engineering of B lymphocytes is our incomplete understanding of the environmental cues that support B lymphopoiesis. Much attention has been afforded the identification of soluble factors that promote B lymphopoiesis ex vivo, but the requirement for stromal cells remains less well understood. In preliminary work we have made the unexpected observation that non-cell-autonomous Hh signaling in stromal cells promotes B lymphopoiesis from hematopoietic progenitors. Our inference rests on several observations: (1) Hh signaling contributes to the maintenance of B progenitors; (2) cell-autonomous Hh signaling is dispensable for B cell development and function; and (3) B lymphopoiesis is impaired by extinction of Hh signaling in stromal cells, but not in hematopoietic progenitors. Depletion of Smo from stromal cells is associated with coordinate downregulation of genes associated with osteoblastoid identity and B lymphopoietic activity, leading us to propose that Hh pathway activity maintains a stromal phenotype that promotes early B lymphopoiesis. The work proposed under this proposal aims to test this hypothesis, and in so doing to develop deeper mechanistic understanding of non-cell-autonomous signals in B cell development. Under the first aim we will define the developmental block to B lymphopoiesis upon extinction of Hh signaling in stromal cell lines and in primary stroma. In the second aim we will exploit biochemical and in silico methods, extensive genomic resources and robust biological systems to identify and validate novel, Hh-dependent stromal factors that pro- mote B lymphopoiesis in the mouse and human. This goal is of particular importance as it is expected to un- cover new mechanisms by which stromal cells instruct early hematopoiesis and new tools for the engineering of immune responses. The third aim will define the effects of non-cell-autonomous Hh signaling on lymphoid development in vivo through the use of mouse models for manipulation of Hh signaling in osteoblastoid stroma. Thus the overarching theme of this proposal is to define the contribution of the Hh morphogenetic pathway to B cell development. Accomplishment of these aims would enable the construction of robust systems for B lymphoid differentiation from hematopoietic progenitors, thereby overcoming a critical roadblock in the engineering of humoral immunotherapies.